Expression of bacterial virulence genes often correlates with the exhaustion of nutrients, but how the signaling of nutrient availability and the resulting physiological responses are co- ordinated is unclear. Until this gap in knowledge is closed, metabolically diverse bacteria like Staphylococcus aureus will continue to cause perilous hospital-acquired infections. The applicant's long-term goal is to lead an independent academic research group studying how bacteria integrate and respond to information provided by intracellular metabolites (the metabolome) to reconfigure metabolism to adapt to environmental changes and cause disease. The objective of this project is to augment existing genetic and biochemical expertise with high- throughput global techniques to analyze gene expression, intracellular metabolites and flux, and, in doing so, titrate the activity of the global regulator CodY and deduce its regulatory hierarchy in S. aureus. At the heart of this project is the hypothesis that fluctuations in the intracellular pools of branched-chain amino acids and GTP result in a spectrum of CodY activities that produce a graded response to nutrient limitation, culminating in metabolic adaptation and the development of virulence. This hypothesis is based on preliminary studies that identified the true intracellular metabolites that control CodY activit in living cells and revealed hierarchical organization for three genes. The rationale for this project is that comprehensive knowledge of the co-regulation of metabolism and virulence is essential if we are to understand the physiological origins of bacterial pathogenesis. During the mentored (K99) phase at Tufts University School of Medicine, massively parallel sequencing, mass spectrometry-based metabolomics and chemostat cultivation will be mastered to map intersecting metabolic and virulence gene expression patterns in S. aureus, while gaining critical scholarly training needed to launch a successful independent academic career with guidance from a mentoring committee composed of experts in bacterial physiology, biochemistry and systems biology. Mastering the cultivation and genetic manipulation of pathogenic S. aureus along with high-throughput methods will enable efforts during the R00 phase to quantify changes in the S. aureus CodY regulon upon induction of physiological stress response systems. The approach is innovative, because continuous bacterial cultures mimic nutrient-limiting bacterial niches in the human body and the experiments will place virulence gene expression in the context of the normal behavior of S. aureus under the nutrient-limiting conditions of the host. Furthermore, correlations between global metabolite pools and CodY activity will provide a previously unattainable linkage of the transcriptome to the metabolome. The project is significant because it will increase our understanding of how the genetic pro- grams of metabolic adaptation and virulence gene expression are interrelated and interdependent. A more thorough understanding of these connections may also offer potentially novel therapeutic strategies. The Pathway to Independence Award will provide the time and resources needed to achieve these goals. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because understanding how bacteria connect metabolism and disease-causing processes may reveal new ways to prevent the switch from harmless to harmful lifestyles that lead to potentially life-threatening infections. The research and career development proposed in this Pathway to Independence award application will unravel cellular mechanisms underlying bacterial disease while providing a stepping stone to an independent academic career - objectives that align with the mission of the National Institute of General Medical Sciences.